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Abstract:

The present invention relates to a fibroblast activator comprising grape
sap as an active ingredient, a method of activating a fibroblast, using
grape sap, and a method of using grape sap as a fibroblast activator. The
present invention further relates to a collagen synthesis promoter
comprising grape sap as an active ingredient, a method of promoting
collagen synthesis using grape sap, and a method of using grape sap as a
collagen synthesis promoter. The present invention further relates to a
skin antiaging agent comprising grape sap as an active ingredient, a
method of preventing aging of skin using grape sap, and a method of using
grape sap as a skin antiaging agent. The present invention provides
highly safe and effective means of preventing aging.

14. The skin antiaging agent according to claim 13, further comprising
proline.

15. A method of preventing aging of skin, using grape sap.

16. The method of preventing aging of skin, using proline together.

17. A method of using grape sap as a skin antiaging agent.

18. The method according to claim 17, wherein the skin antiaging agent
further comprises proline.

Description:

TECHNICAL FIELD

[0001]The present invention relates to a fibroblast activator that
activates fibroblasts participating in the decomposition and synthesis of
collagen, a method of activating a fibroblast, a collagen synthesis
promoter, a method of promoting collagen synthesis, a skin antiaging
agent capable of preventing aging of skin by activating fibroblasts and
promoting collagen synthesis, and a method of skin antiaging.

BACKGROUND TECHNIQUE

[0002]Generally, aging of skin is the physiological phenomenon that is
produced by the combined effects of physiological aging with the
advancement of age and photoaging caused by ultraviolet radiation. Upon
entering one's 20's or 30's, proteins in the skin, such as the collagen
that is the source of skin elasticity in the dermal layer, decrease with
age. The strength of the collagen-producing fibroblasts themselves also
decreases. Photoaging is a phenomenon whereby enzymes that decompose
collagen are secreted due to exposure to ultraviolet radiation of the
skin, thereby damaging the collagen and causing loss of dermal
elasticity. Such reduction in the quantity of collagen and diminished
functioning of fibroblasts are major causes of aging phenomena such as
wrinkling and sagging.

[0004]However, by simply inhibiting the activation of collagen decomposing
enzyme, merely collagen decomposition by ultraviolet radiation
(photoaging) is prevented, but skin aging that has undergone advanced due
to ultraviolet radiation or increasing age cannot be effectively
improved.

[0005]Retinolic acid (retinoic acid, a derivative of vitamin A) is well
known as an antiaging component that rather effectively promotes the
synthesis of collagen itself (Methods in Enzymology, 1990, vol. 190, p.
352-360).

[0006]However, retinolic acid is strongly irritating, and it is limited to
use by doctors in Japan. Thus, there is need for the development of a
highly effective and safe antiaging agent.

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

[0007]Under these circumstances, it is an object of the present invention
to provide a highly safe and effective means of preventing aging.

Means for Solving Problems

[0008]The present inventors conducted extensive research into achieving
the above-stated object, resulting in the discovery that grape sap has a
fibroblast-activating function and a function of promoting collagen
synthesis by fibroblasts; the present invention was devised on this
basis.

[0009]The present invention relates to a fibroblast activator comprising
grape sap as an active ingredient, a method of activating a fibroblast
using grape sap, and a method of using grape sap as a fibroblast
activator.

[0010]The present invention further relates to a collagen synthesis
promoter comprising grape sap as an active ingredient, a method of
promoting collagen synthesis using grape sap, and a method of using grape
sap as a collagen synthesis promoter.

[0011]The present invention further relates to a skin antiaging agent
comprising grape sap as an active ingredient, a method of preventing
aging of skin using grape sap, and a method of using grape sap as a skin
antiaging agent.

EFFECTS OF THE INVENTION

[0012]According to the present invention, aging of the skin can be
effectively prevented by promoting the synthesis of collagen. Further,
when proline is used in combination with grape sap, the present invention
can further strengthen the effect of grape sap.

BEST MODE FOR CARRYING OUT THE INVENTION

[0013]The present invention will be described in more detail below.

[0014]The present invention relates to a fibroblast activator comprising
grape sap as an active ingredient, and the fibroblast activator of the
present invention can comprise proline in addition to grape sap.

[0015]The present invention relates to a method of activating a fibroblast
using grape sap, and the method of activating a fibroblast of the present
invention can use proline in addition to grape sap.

[0016]The present invention relates to a method of using grape sap as a
fibroblast activator, and in the method of using of the present
invention, the fibroblast activator can comprise proline in addition to
grape sap.

[0017]Collagen is a fibrous protein present in almost all cells, such as
skin, blood vessels, tendons and teeth, accounting for about 30 percent
of all protein constituting the human body. The human body is comprised
of equal to or more than 60 trillion cells. Cells and organs comprised of
cells are properly shaped by the extraceilular matrix. Collagen is the
protein that makes up the extracellular matrix. It serves as a
scaffolding for cells, plays roles in the proliferation and functional
maintenance of cells, and plays important roles as a constituent
component of matrix proteins in cartilage and bone.

[0018]Fibroblasts are one of the main types of cells that produce
collagen. The collagen that is secreted by fibroblasts fills in the gaps
between cells to form the intercellular matrix. As a result of
investigation, the present inventors discovered for the first time that
grape sap has a function of activating fibroblasts themselves (a
fibroblast-activating function).

[0019]The use of proline in combination with grape sap can enhance the
fibroblast-activating effect possessed by grape sap. The proline is
preferably L-proline.

[0020]The present invention relates to a collagen synthesis promoter
comprising grape sap as an active ingredient, and the collagen synthesis
promoter of the present invention can comprise proline in addition to
grape sap. The present invention relates to a method of promoting
collagen synthesis using grape sap, and the method of promoting collagen
synthesis of the present invention can use proline in addition to grape
sap. The present invention relates to a method of using grape sap as a
collagen synthesis promoter, and in the method of using of the present
invention, the collagen synthesis promoter can comprise proline in
addition to grape sap.

[0021]As set forth above, grape sap has a fibroblast-activating function.
The present inventors conducted research resulting in the new discovery
that grape sap has the function of promoting collagen synthesis in
fibroblasts. The collagen synthesis promoter of the present invention
comprises, as an active ingredient, grape sap that works on fibroblasts
themselves and has the functions of activating the cells themselves and
promoting collagen synthesis by fibroblasts, thereby effectively
promoting collagen synthesis.

[0022]The use of proline in combination with grape sap can enhance the
collagen synthesis promoting effect possessed by grape sap. The proline
is preferably L-proline.

[0023]The present invention relates to a skin antiaging agent comprising
grape sap as an active ingredient, and the skin antiaging agent of the
present invention can comprise proline in addition to grape sap. The
present invention relates to a method of preventing aging of skin using
grape sap, and the method of preventing aging of skin of the present
invention can use proline in addition to grape sap. The present invention
relates to a method of using grape sap as a skin antiaging agent, and in
the method of using of the present invention, the skin antiaging agent
can comprise proline in addition to grape sap.

[0024]Changes of properties and conditions of collagen, and functions of
fibroblasts are significantly involved with aging of skin, such as loss
of skin tension and wrinkles. This is because, excluding water, the skin
is comprised of roughly 80 percent collagen. The skin is divided into the
three layers of, from the surface down, the epidermis, dermis, and
subdermal tissue. Among them, collagen is present in the dermis,
constituting the greater portion thereof, and has the structure and
properties of a textile. Since various forces are applied to the skin
from the exterior, the skin is required to be strong and flexible; such
properties are mainly due to the functions of collagen. The decomposition
and synthesis of collagen are controlled by the fibroblasts, and collagen
is maintained in a constant state of tension by the strength of the
fibroblasts. The skin antiaging agent of the present invention comprises
grape sap as an active ingredient, and its collagen synthesis promoting
function and fibroblast activating function can effectively prevent aging
of the skin in the form of loss of skin tension and wrinkles.

[0025]The use of proline in combination with grape sap can enhance the
antiaging effect on skin possessed by grape sap. The proline is
preferably L-proline.

[0026]Grape sap is tracheary water that flows in the grape plant (family
Vitis). The type of grape sap employed in the present invention is not
specifically limited other than that it be sap of a plant of family
Vitis; the sap of the grape, Vitis coignetiae, or the like may be
employed. Examples are grape varieties such as: Kyoho, Koshu, Kaiji,
Muscat Berry A, Delaware, Cambell Early, Pione, Neo Muscat, Niagara,
Concord, Steuben, Cabernet Sauvignon, Pinot Noir, Merlot, Semillon,
Riesling, and Chardonnay. Neither the region of cultivation, method of
cultivation, nor the like is limited. However, sap flowing out of the
pruned branches of grape in early spring, before the budding season, is
preferable.

[0027]Grape sap can be obtained by cutting the stems of grape and
collecting the sap that flows out. The method of collecting the sap is
not specifically limited. For example, small containers or the like can
be attached at the sites of pruning so that the sap flows into them, the
sap that flows out can be collected, and these can be collected in a
large storage container to which preservatives or the like have been
added. The use of a device capable of collecting sap from various
locations at once, such as the collection device described in Japanese
Unexamined Patent Publication (KOKAI) Heisei No. 10-295207, and the
liquid receiving container described in Japanese Unexamined Patent
Publication (KOKAI) No. 2005-118035, for collection is preferable because
such collection is highly efficient and deterioration in quality is
prevented.

[0028]As needed, the collected grape sap can be mixed with preservatives
or the like and employed in liquid form or as a freeze-dried product. As
needed, the collected grape sap can be subjected to a heat or
clarification treatment. This prevents the sap from clouding or turning
brown. The heat treatment can be conducted by heating the grape sap to,
for example, 60 to 100° C., preferably 80 to 90° C., for
equal to or longer than 10 minutes, for example. The heat treatment can
be conducted on the grape sap after collection, or conducted following
processing as a product. The clarification treatment can be conducted
using a known clarifying agent, such as activated carbon, bentonite,
silica gel, silica sol, or PVPP (polyvinyl polypyrrolidone).

[0029]The fibroblast activator, collagen synthesis promoter, and skin
antiaging agent of the present invention each can contain grape sap in
liquid form or in the form of a freeze-dried product. The formulation is
not specifically limited. They can be provided in various formulations
such as lotions, emulsions, gels, creams, ointments, powders, and grains.
The fibroblast activator, collagen synthesis promoter, and skin antiaging
agent of the present invention may each be comprised of just grape sap,
or of grape sap and proline. As needed, oil-based components,
surfactants, moisturizers, pigments, ultraviolet radiation-absorbing
agents, antioxidants, fragrance materials, dyes, antibacterial agents,
fungus-combating agents, alcohols, water and the like, that are commonly
blended into pharmaceuticals, quasi-drugs, skin cosmetics and the like,
may be suitably blended in. To the extent that the effect of the present
invention is not lost, other cell activators, collagen synthesis
promoters, and antiaging agents may also be used together.

[0030]The fibroblast activator, collagen synthesis promoter, and skin
antiaging agent of the present invention can comprise grape sap in a
proportion of equal to or greater than 1 weight percent, preferably 3
weight percent to 100 weight percent, and more preferably, 10 weight
percent to 80 weight percent, based on the unadjusted sap. In the present
invention, grape sap itself may be used as a fibroblast activator,
collagen synthesis promoter, and skin antiaging agent.

[0031]When proline is blended into the fibroblast activator, collagen
synthesis promoter, or skin antiaging agent of the present invention in
addition to grape sap, the quantity blended in suitably falls within a
range of 0.1 to 3 weight percent, preferably 0.1 to 1.5 weight percent.
The quantity of proline blended in is given relative to the total of
proline and grape sap being 100 percent.

[0032]The method of applying the fibroblast activator, collagen synthesis
promoter, or skin antiaging agent of the present invention as a topical
agent to the skin may be suitably selected based on the type of topical
agent. Generally, when applying it as a topical agent prepared as a
lotion, emulsion, gel, cream, or ointment, approximately one or two
applications daily to the facial skin or the like is preferable. The
fibroblast activator, collagen synthesis promoter, and skin antiaging
agent of the present invention can be employed as cosmetics, and can be
applied to lotions, emulsions, beauty solutions, creams, liquid
foundations, powder foundations, lipstick, and the like.

[0033]The quantity of the fibroblast activator, collagen synthesis
promoter, or skin antiaging agent of the present invention administered
when employed as an orally administered drug in the form of a powder or
grain can be suitably selected based on conditions such as the age and
skin condition of the person to whom it is being administered. The
quantity administered falls within a range of, for example, 100 to 1,000
mg (as a quantity of active ingredient) for an adult. However, the
above-stated administration quantity can be suitably altered based on the
above-stated conditions.

EXAMPLES

[0034]The present invention will be described in greater detail below
through Examples. However, the present invention is not limited to
Examples given below.

[0035]The grape sap employed in Examples below was obtained by using the
device described in Japanese Unexamined Patent Publication (KOKAI) Heisei
No. 10-295207 to collect sap running out of grape (variety: Koshu)
branches being trimmed in a vineyard in Katsunuma-cho, Higashi
Yamanashi-gun, Yamanashi Prefecture in early April, 2005. One gram of
freeze-dried grape sap was equivalent to 280 g of unadjusted sap.

Example 1

Test of Fibroblast Activation Function

[0036]3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromate
(MTT) cleaves NADPH or NADH produced in energy metabolic processes in
mitochondria within cells to produce formazan. Thus, the quantity of
formazan produced was adopted as an indicator of cell activation
function.

[0037]Normal human fibroblasts were seeded onto a 96-well microplate at a
cell density of 3.0×104 cells/well. After culturing for 24
hours in Dulbecco's modified eagle medium (DMEM) containing 1 percent
fetal bovine serum (FBS), the medium was replaced with DMEM containing 1
percent FBS containing a freeze-dried product of grape sap in the
concentration shown in Table 1. A positive control (PC) in the form of
DMEM containing 5 percent FBS was employed. After culturing for 48 hours,
the medium was replaced with DMEM containing 1 percent FBS containing 0.4
mg/mL MTT (Nacalai) and cultivation was conducted for two hours.

[0038]The cells were washed, the formazan produced in the cells was
dissolved in 2-propanol, and a microplate reader was employed to measure
the absorbance at 550 nm and 650 nm. The difference between the two was
calculated to evaluate the quantity of formazan generated within the
cells. Cell activation function was denoted by an activation index (%)
with the absorbance (ABS.) of cultivated cells to which no sample was
added (control) being denoted as 100. The results are shown in Table 1.
All the results are given as the average value±standard deviation.
Student's t-test was employed to determine significant differences. The p
value was determined to be significant when p<0.05. As shown in Table
1, cultivation in a medium to which grape sap had been added was
determined to activate fibroblasts.

[0039]The collagen synthesis of fibroblasts was evaluated by quantifying
the amount of secreted type I collagen in culture supernatant by ELISA.

[0040]Normal human fibroblasts were seeded onto a 96-well microplate at a
cell density of 3.0×104 cells/well. After culturing for 24
hours in DMEM containing 0.5 percent FBS, the medium was replaced with
DMEM containing 0.5 percent FBS containing a freeze-dried product of
grape sap in the concentration shown in Table 2. A PC in the form of
magnesium ascorbyl phosphate (VCPMg) was employed. After 48 hours, the
culture supernatant was collected and the amount of type I collagen was
quantified by ELISA.

[0041]Culture supernatant and calibration curve-use type I collagen were
placed in a 96-well plate and coated overnight at 4° C. A 1
percent bovine serum albumin (BSA) solution was used to conduct blocking
for 1 hour at 37° C., after which the solution was reacted for 1.5
hours at 37° C. with primary antibody (antihuman type I collagen
(rabbit)). It was then reacted for 1.5 hours at 37° C. with
secondary antibody (Histofine PO (rabbit)), after which phosphate-citrate
buffer (0.1 M, pH 4.0) containing 0.3 mg/mL of
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate)diammonium (ABTS) was
added. Twenty minutes later, a microplate reader was employed to measure
the absorbance at 405 nm. The quantity of collagen in the culture
supernatant was calculated from a calibration curve measured with the
same plate.

[0042]At the same time, the quantity of cell protein was quantified using
BCA protein assay reagent (PIERCE). The quantity of collagen in the
culture supernatant was divided by the quantity of protein to calculate
the quantity of collagen per unit of protein, which was adopted as the
quantity of collagen synthesized by the cells. The results are given in
Table 2. All of the results are denoted as the average value±standard
deviation. Student's t-test was employed to determine significant
differences. The p value was determined to be significant when p<0.05.
As shown in Table 2, cultivation in a medium containing grape sap was
found to promote collagen synthesis by fibroblasts by about 1.2-fold to
3.6-fold that when grape sap was not added (control). The function was
particularly marked when medium containing a freeze-dried product of
grape sap of equal to or greater than 500 micrograms/mL was employed.
When medium containing a freeze-dried product of grape sap of equal to or
greater than 250 micrograms/mL was employed, a significant increase in
protein quantity was observed. The increase in protein indicated that the
fibroblasts had been activated and had proliferated. The quantity of
collagen shown in Table 2 is a value that has been divided by the amount
of protein, and it can be understood that the collagen synthesis levels
of individual cells increased significantly.

[0044]To determine the effects of using proline in combination with grape
sap, a cream was formulated as indicated in Table 3 for use in the
following organoleptic evaluation tests and wrinkle evaluation tests. All
blending quantities of the compositions given in Table 3 are weight
percentages.

[0046]Sixty women aged from 30's to 50's who stated that their skins were
dry or lacked suppleness or tension were employed as test subjects. They
were divided into four groups of 15 each without deviation in age. The
creams of the four formula examples given in Comparative Example 1 and
Examples 1 to 3 indicated in Table 3 were given to the respective groups
of test subjects, who were requested to use them twice a day, once in the
morning and once at night, for 30 consecutive days. An organoleptic
evaluation test was conducted on the use sensations: a moist sensation
(moisturizing property), tension sensation (resilience), and softness
sensation (flexibility). The evaluation criteria are given in Table 4,
and the results of the organoleptic evaluation test in Table 5.

[0048]Forty women aged from 40's to 60's who were concerned about skin
wrinkles (particularly small wrinkles at the corner of the eye) were
employed as test subjects. They were divided into four groups of 10 each
without deviation in age. The creams of the four formula examples given
in Comparative Example 1 and Examples 1 to 3 indicated in Table 3 were
given to the respective groups of test subjects, who were requested to
use them twice a day, once in the morning and once at night, for 30
consecutive days while focusing on the areas around the eyes where
wrinkles tend to form. The numbers of wrinkles at the corners of the eyes
before and after application were compared by visual determination using
UV photography. The results are given in Table 6.

[0050]Both in Table 5 showing the organoleptic evaluation test results and
in Table 6 showing the wrinkle evaluation test results, the cream
containing grape sap of Example 1 afforded better effects in terms of
improvement in dryness, tension, wrinkles, and the like than the cream
containing proline of Comparative Example 1. Further, the creams of
Examples 2 and 3, containing combinations of grape sap and proline,
afforded better effects in terms of improving dryness, tension, wrinkles,
and the like than either grape sap or proline employed alone. That is,
grape sap has an excellent effect in terms of improvement in dryness,
tension, wrinkles, and the like. Further, the combining amino acids that
constitute collagen, particularly proline, with grape sap for use is
found to enhance the moisturizing effect on skin and further enhance the
effect of reducing the appearance of wrinkles.

INDUSTRIAL APPLICABILITY

[0051]The present invention can effectively prevent aging of the skin
through the fibroblast activation function of grape sap and through the
collagen synthesis promoting function of grape sap on fibroblasts.

[0052]In addition to preventing aging, the application of the collagen
synthesis promoter and the fibroblast activator of the present invention
can be anticipated for use on the treatment of pimple marks and pregnancy
stretch marks, the treatment of wounds such as burn wounds, promoting
artificial skin cultures, and the like.